Session 7: Evolution of Vertebrates

Transcript of Part 3: The Evolution of Limbs from Fins

[00:00:07.15]My name is Neil Shubin from the University of Chicago,
[00:00:09.25]and we're going to talk today
[00:00:11.07]about organogenesis in deep time.
[00:00:13.27] In particular, we're going to look at this:
[00:00:15.15]we're going to try to compare a fish to a human.
[00:00:17.10]How do you compare a fish fin
[00:00:19.25] to a human limb?
[00:00:21.02]How did the limb come about from fins?
[00:00:23.00] And what are the different ways we pull together
[00:00:25.26] different types of data?
[00:00:27.20]Let's think of it this way,
[00:00:29.04]this is a nice starting point.
[00:00:31.11] Think about comparing, like I say,
[00:00:33.05]a human arm to a fin of a fish,
[00:00:34.27] shown on the left here.
[00:00:36.14]They look very different.
[00:00:38.01] Right, if you look at the bones, shown in black,
[00:00:39.25]there doesn't really seem to be a whole lot of correspondence.
[00:00:43.02]Fish have lots of bones,
[00:00:44.11]we have the one bone, two bone, little bone, finger pattern
[00:00:47.10] seen in chickens and whales and everything with limbs.
[00:00:50.22]Also, fish have fine rays, which we don't have.
[00:00:53.12]Big differences,
[00:00:56.18]yet we can bridge these gaps when we look at fossils.
[00:00:59.12] If we were to fill this diagram with some of the fossils,
[00:01:01.29]what you see here are
[00:01:04.19]you start to see lots of finned creatures,
[00:01:06.27]creatures with fin webbing, fin rays,
[00:01:09.25]but also having the one bone, two bone, little bone pattern as well.
[00:01:15.07]So what this means is, if we want to bridge the gap
[00:01:17.10] between fins and limbs,
[00:01:19.02]what we need to do is
[00:01:21.12]to have expeditions targeted to key parts of the tree of life.
[00:01:25.10]That is, we can target certain time periods to find fossils,
[00:01:28.18]and some of those fossils
[00:01:30.10]will start to bridge the gap between fins and limbs.
[00:01:34.25] But that's not the only thing that's important here.
[00:01:37.01] When we start to have these fossils,
[00:01:39.00] we can start to compare
[00:01:41.02]living creatures in different ways.
[00:01:42.09] That is, we can start to compare
[00:01:43.22] a human arm
[00:01:45.14]to the fin of a fish
[00:01:47.06]by seeing correspondences
[00:01:49.27]that would have been absent to us without the fossil evidence.
[00:01:52.01]What that enables us to do is to design experiments,
[00:01:54.24] that is, we can design new experiments
[00:01:56.18] based on our paleontological understandings
[00:01:59.00]on the developmental genetics of all kinds of different kinds of fish,
[00:02:02.12] non-model organisms.
[00:02:04.24]And in fact it works all ways.
[00:02:06.04]Once we have these experiments
[00:02:08.04] based on non-model organisms,
[00:02:09.20]we can begin to target new parts of the tree of life
[00:02:11.22]where we may be missing fossil data.
[00:02:14.01]So, the central idea here is
[00:02:16.25]that fossils enable us to bridge gaps in the record,
[00:02:21.05]the anatomical record of the tree of life,
[00:02:23.23]that enables us to design experiments in developmental genetics
[00:02:27.05] on living creatures,
[00:02:28.06]and the more we understand about developmental genetics of living creatures,
[00:02:30.15] the more we understand about what gaps exist in the fossil record
[00:02:33.17]and where we need to lead the next expeditions.
[00:02:36.01] So, really, the fossil and genetic data
[00:02:38.11]work hand in hand.
[00:02:39.21]So, let's work through an example here.
[00:02:42.21]Well, our work obviously begins with the origin of tetrapods,
[00:02:45.03]the transition, say,
[00:02:46.15]of something like a fish on top
[00:02:48.03]to a limbed animal on the bottom,
[00:02:50.16]and we can design expeditions that bridge this gap,
[00:02:54.05]and what we do is we look for places in the world
[00:02:56.27] that have rocks of the right age,
[00:02:58.16]rocks of the right type,
[00:03:00.03]and rocks that are exposed to the surface
[00:03:02.22] for us to find fossils.
[00:03:03.20]Using that tool kit, we can begin to bridge this gap.
[00:03:07.00]It turns out to understand the origin of tetrapods
[00:03:08.29]we need to focus on environments like this,
[00:03:11.06] near-shore marine environments like ancient seaways,
[00:03:13.25]but likewise ancient highlands,
[00:03:16.10]so delta systems turn out to be really perfect for us,
[00:03:20.14]because when we have a system like this
[00:03:22.12]we can sample ancient seas,
[00:03:23.29]ancient estuaries,
[00:03:25.11]ancient rivers and streams,
[00:03:26.20]you know, the whole enchilada, as they say.
[00:03:29.14] So, really, it became clear
[00:03:31.29]very early in our study
[00:03:34.13]that the best places that had these kind of delta systems
[00:03:36.18]of the right age, in the late Devonian period,
[00:03:39.14]were centered in three general places in North America
[00:03:42.07] -- this is a slide actually taken from an undergraduate college geology textbook,
[00:03:46.13] which helped us launch a number of expeditions --
[00:03:48.27]but it became very clear to us that
[00:03:50.27] two of these areas that were seen in this diagram
[00:03:52.10]were known by scientists before.
[00:03:54.18]We had previously worked
[00:03:56.21]on the so-called Catskill rocks of eastern Pennsylvania.
[00:03:59.10]Other colleagues had worked in East Greenland,
[00:04:02.18]these are very well studied rocks.
[00:04:04.16]If in this diagram you can see what led us to the Arctic in the first place
[00:04:08.07] is rocks of the right age, rocks of the right type,
[00:04:09.20] rocks exposed across the surface,
[00:04:11.20]in an area of the Arctic
[00:04:14.14] that was completed explored by vertebrate paleontologists.
[00:04:16.28]So we had ideal geology,
[00:04:18.18]but really very few of our colleagues had worked on these rocks.
[00:04:21.05] So off we went.
[00:04:23.16]Anytime you talk about a fossil expedition
[00:04:25.06]you're talking about teamwork,
[00:04:26.22]and I just want to give credit where credit's due.
[00:04:28.01]My graduate mentor, Ferris Jenkins,
[00:04:30.00]he and I have these big smiles on our faces
[00:04:32.00] -- the reason why is something that's in this plaster jacket, here.
[00:04:35.18]My good friend and colleague Ted Daeschler,
[00:04:38.03]shown in the upper left,
[00:04:39.07]he's been a partner in these expeditions for decades.
[00:04:41.24] Likewise, all the field crews
[00:04:43.23]that we've had over several decades
[00:04:45.04]as well as the lab team as well.
[00:04:46.10]This is a team effort, discovering fossils.
[00:04:48.18]We don't go out there alone,
[00:04:50.02]we go out there in teams of very talented people.
[00:04:52.20]So, we started these expeditions in 1999,
[00:04:56.28]based on this kind of map,
[00:04:58.21] and what you see on this map
[00:05:00.12]are the islands of the Canadian Arctic,
[00:05:03.05]and surrounded in red are where the Devonian-age rocks are exposed.
[00:05:07.06] And the first set of them that we did,
[00:05:08.18]we had to get to by these helicopters and planes
[00:05:11.09] because it's pretty far away.
[00:05:12.28] This sort of dictates the kind of science that we can do.
[00:05:16.10]Using this, spending several hours
[00:05:19.01] on a helicopter or a plane,
[00:05:20.07]we got to the western part of the Canadian Arctic,
[00:05:22.24]shown on the arrow here.
[00:05:25.01]This area was, you know, ideal for exposures.
[00:05:27.04] What you see is a vista,
[00:05:29.10]a plain of Devonian-age rocks
[00:05:31.20]all across this landscape, here,
[00:05:33.27]but this was the wrong fossil environment
[00:05:36.20]to hold the kinds of creatures we were interested in.
[00:05:39.11] This was an ancient marine system,
[00:05:41.08] this was a system that had ancient deep-water sediments,
[00:05:44.27]so we weren't finding the kind of critters we were on the hunt for,
[00:05:48.15] which is, say, a flat-headed fish with fins.
[00:05:50.20]So we had to retool a little bit,
[00:05:52.14]so we used the geological understandings here.
[00:05:54.24]This is an ancient delta system...
[00:05:56.20]we were in the ancient seaway...
[00:05:58.27]we needed to move upstream.
[00:06:00.16]To move upstream in the ancient geological rocks
[00:06:03.20] meant moving east.
[00:06:05.00]So, we went east, and then the next year,
[00:06:07.05]you can see here in 2000, where the arrow is,
[00:06:09.19]we went to southern Ellesmere Island.
[00:06:11.00]This is what it looked like; it's a really marvelous place.
[00:06:13.16]Moraine with, you know, with red rocks.
[00:06:15.18]This contained ancient rivers and streams
[00:06:18.17] that held a number of lobe-finned fish critters.
[00:06:22.09]We homed in on a particular valley
[00:06:23.27] that had a layer of fossil fish
[00:06:27.09]that were preserved one on top of the other.
[00:06:29.20] These fish were very well preserved
[00:06:32.06] and it really wasn't until 2004
[00:06:34.09] that one of my colleagues removed a rock from this layer...
[00:06:37.16] Steve Gates, who is a professor at Brown University here on the left,
[00:06:41.06]removed a rock here and he saw a V.
[00:06:43.24] And he called us over and he said, "What's this bone here?"
[00:06:45.28] You can barely see it in this picture,
[00:06:47.29]but it was beautiful,
[00:06:49.25]because what it is is it's a snout of a fish,
[00:06:52.11]and not just any fish,
[00:06:54.07]it's a snout of a flat-headed fish.
[00:06:55.18]And one of the big transitions
[00:06:57.08]is going from a conical head to a flat-headed animal.
[00:06:59.05] Here I had a flat-headed fish looking right at me.
[00:07:02.03]So we bring these things move,
[00:07:03.18] it turns out we found four of them this first year,
[00:07:05.22] they come home and the preparers begin to work on them,
[00:07:08.22]removing the rock grain by grain,
[00:07:11.06]and you could see what's emerging here
[00:07:13.02] is a flat head with eyes on top.
[00:07:15.14]Several months later, you could see this thing exposing even more.
[00:07:18.08]You could see the head revealing itself.
[00:07:20.15]You can even see the shoulder girdle, here,
[00:07:23.06]revealing itself,
[00:07:24.25]and maybe this creature even has a neck.
[00:07:27.02] Remember what this quest is all about:
[00:07:28.29]sort of bridging this gap
[00:07:30.20]between lobe-finned fish and a limbed animal.
[00:07:33.22]Maybe finding a flat-headed fish with fins...
[00:07:35.23] this is what the expedition led to,
[00:07:37.13]a flat-headed fish with fins.
[00:07:39.02]Like a fish, it has scales on its back
[00:07:41.16] and fins with fin webbing.
[00:07:43.09]Like a tetrapod, it has a flat head
[00:07:45.09]with eyes on top, a neck,
[00:07:47.15]and, when we cracked open the fin, w
[00:07:49.15]e found bones that correspond to upper arm, forearm,
[00:07:52.02]even parts of a wrist.
[00:07:54.10] Here's a CT scan of the fin
[00:07:56.24]and you can see what it has is a humerus,
[00:07:58.09]and then two bones here,
[00:07:59.22]a radius and an ulna,
[00:08:01.06]and shown in blue are the fin rays,
[00:08:03.06]so it's a real mix of characteristics.
[00:08:05.10]Shown on the left, this is the work of Justin Lemberg,
[00:08:09.01]a graduate student in my laboratory,
[00:08:10.10]shows the joints of this animal.
[00:08:12.10]In a you see the shoulder of this animal,
[00:08:15.25] the socket of the shoulder on the left
[00:08:18.00]and the ball of the humerus on the right.
[00:08:20.23] This is a fish with an elbow, you can see the elbow in b,
[00:08:23.27]and there are even two parts of a wrist,
[00:08:25.16] a proximal carpus and a distal carpus.
[00:08:28.12] This is a fish with components
[00:08:30.12]of our own anatomy inside.
[00:08:32.16]And we can use CT scanning,
[00:08:34.09]as you can see in the image here...
[00:08:37.07]we can begin to dissect the skull using CT scanning
[00:08:40.14]and begin to see the individuals bones
[00:08:42.23]and how they suture together.
[00:08:44.29] It turns out that when we use living animals...
[00:08:47.10]this is an alligator gar shown on the bottom right, here,
[00:08:49.19]and the alligator gar will bite animals in the water,
[00:08:52.10]but as it does so the bones of the skull
[00:08:55.02]show cranial kinesis.
[00:08:56.20]They move in particular ways relative to one another,
[00:09:00.00]and when we analyze Tiktaalik's skull, here,
[00:09:02.19]which is this fossil creature I'm showing you,
[00:09:04.25]we can begin to see that the joints of this animal's skull
[00:09:07.22] can actually move.
[00:09:09.10] It has cranial kinesis,
[00:09:10.25]much like a living alligator gar.
[00:09:12.28] So, what I'm saying is when we find these fossils,
[00:09:14.28]the discovery is really only the beginning,
[00:09:17.11]because then we can start to work on their anatomy,
[00:09:20.23] compare them to other creatures,
[00:09:22.07]and begin to assess their biomechanics
[00:09:24.01] -- how they ate, how they walked,
[00:09:25.26]and how they lived in these aquatic environments,
[00:09:31.03] in Devonian streams.
[00:09:32.11]So, we have this creature, Tiktaalik roseae,
[00:09:33.24]it's an animal that has lungs and gills,
[00:09:35.25] it has fins that have components of limbs inside,
[00:09:38.06]it has a neck...
[00:09:40.24]it really has a mix of characteristics.
[00:09:42.07] And when we map this in the phylogenetic tree,
[00:09:45.06]what we see is it holds a relatively special place.
[00:09:48.16]That is, you can see the fish on the bottom
[00:09:50.18]and the limbed animals on top.
[00:09:52.12]Tiktaalik sits right here in the middle.
[00:09:54.20]It shows us the sequence
[00:09:56.25]of the acquisition of tetrapod characteristics,
[00:10:00.05] whether it's necks, fingers, wrists, toes,
[00:10:03.09]and so forth.
[00:10:05.09] Well, how is this relevant to developmental biology?
[00:10:07.17] Well, remember what we're saying
[00:10:10.10]is when we have fossils like Tiktaalik
[00:10:12.02] we can compare the arms of, say,
[00:10:14.04]people and chickens and mice
[00:10:16.10]to the fins of fish in novel ways.
[00:10:18.28]What creatures like Tiktaalik are showing us
[00:10:22.16]is that fish, back in the Devonian, had wrists.
[00:10:25.24] They had components of the distal,
[00:10:29.18]the terminal ends of the appendage,
[00:10:30.23] such as seen in our own limbs.
[00:10:33.06]So what that means is,
[00:10:35.06]if the fossil should be read at face value,
[00:10:36.28]is that sometime in the distant past,
[00:10:38.28]and maybe even in living fish,
[00:10:40.13] there should be the machinery
[00:10:42.16]by which limbs and toes and fingers and wrists and ankles
[00:10:46.07]are developed.
[00:10:48.01]So, let's get back to this comparison here.
[00:10:49.24] If you look at a zebrafish, say,
[00:10:52.24]the fish on the left,
[00:10:54.23]and a human here on the right,
[00:10:56.29] you know, the bones of the fins don't look very similar.
[00:10:59.14]Where the similarities start to emerge
[00:11:02.03]is when we compare them to the fossils,
[00:11:03.16]like I just showed you,
[00:11:04.29]but also when we compare their development.
[00:11:07.02]See, what we have here is a chicken limb in its development,
[00:11:09.23]taken from a textbook,
[00:11:10.29]and you can see the limb bud shown on the left.
[00:11:13.08] It develops this little bud, sticks out of the body,
[00:11:15.12]and as it develops the cartilage skeleton begins to form.
[00:11:20.06]Now, what's driving the development of that cartilage skeleton
[00:11:23.17]are a set of interactions
[00:11:26.00]among signaling centers, like this region here,
[00:11:28.18] the AER, and another one seen at the bottom here,
[00:11:31.26] the ZPA,
[00:11:33.04] but as well as other factors, genes and proteins,
[00:11:34.22]that are turned on and off,
[00:11:36.00]driving the patterns of development
[00:11:37.21]and pattern formation
[00:11:39.14]that are so characteristic of limbs.
[00:11:41.06]Really, the comparison we want to make
[00:11:42.27] is not just between the structures of fins and limbs,
[00:11:45.24]but the developmental mechanisms
[00:11:47.26]by which the skeletal patterns of fins and limbs emerge.
[00:11:51.00]How similar are they and how different are they?
[00:11:53.09]So, to do that,
[00:11:55.19]we focus on a variety of different signaling systems,
[00:11:57.02]as well as transcription factors.
[00:11:58.23] One of the transcription factors that's been incredible important to us
[00:12:02.20] are the Hox genes.
[00:12:03.27]The Hox genes have been shown to be important
[00:12:05.25] in a variety of processes of development
[00:12:07.24]from hindbrains to the axial skeleton to the limbs.
[00:12:10.28] To give you an example of why they're considered so important,
[00:12:13.12]here's a wild type mouse limb.
[00:12:15.22] If you knock out some of the Hox genes
[00:12:18.01] what are known as the 13 paralog groups, Hoxd13 and Hoxa13,
[00:12:22.20]you can develop a mouse limb
[00:12:26.06]that has no fingers, toes, or wrists or ankle bones.
[00:12:29.24]And these are segment-specific modifications of the appendage.
[00:12:33.16]And if you knock out elements
[00:12:36.11] of the 11 cognate groups,
[00:12:37.19]you're missing the middle segment of the appendage.
[00:12:40.13] So, these are genes that are really involved
[00:12:42.12] with the specification of different components of our appendages.
[00:12:46.04]They take a very, very special role
[00:12:48.24]in our understanding of the origin of digits from fish fins.
[00:12:52.22]The question is, how have these patterns of expression
[00:12:56.03]and the patterns of activity
[00:12:57.28]of these genes evolved?
[00:12:59.13]Are they present in fish?
[00:13:00.20]Are they doing similar things in fish?
[00:13:02.00]What's involved in their regulation and their activity?
[00:13:03.20]How is this assembled
[00:13:06.06]going from fish to limbed animals?
[00:13:07.23]Well, there have been a number of studies
[00:13:09.25]of the expression of these genes
[00:13:11.22] in diverse limbed animals,
[00:13:13.01]and interestingly they follow two phases of expression.
[00:13:16.05]Look at the limbed skeleton on the left.
[00:13:19.03]That has three components.
[00:13:20.10]It has a top component consisting of one bone,
[00:13:22.18]it has a central segment composed of two bones,
[00:13:26.19]and then it has a distal segment composed of multiple bones.
[00:13:29.21] It turns out there are two phases in the expression of Hox genes
[00:13:32.14] that are involved with the specification of these components of the appendage.
[00:13:36.05]The earliest phase, shown on top here,
[00:13:38.10]shows the different genes of the Hox system
[00:13:41.19]expressed within one another,
[00:13:44.05]so these are like nested sets of expression
[00:13:46.24]of one set of genes
[00:13:49.13] in the domain of expression of another --
[00:13:51.05]think of Russian dolls.
[00:13:52.29]This phase of expression acts early in limb development
[00:13:55.15]and is involved in the specification
[00:13:57.20] of the first two segments of the appendage.
[00:14:00.28] Coming on later
[00:14:03.05]is a late phase pattern of these same genes.
[00:14:05.24]This involves expression
[00:14:08.08]across the entire distal domain
[00:14:10.04] of what will become the digits and wrist of the limb,
[00:14:13.17]and activity of the late phase
[00:14:16.17]is what's driving specification of the distal component,
[00:14:19.18]that component which includes the wrist
[00:14:22.21]and finger bones of tetrapods.
[00:14:24.18] It's an open question:
[00:14:26.02]To what extent is the origin of the tetrapod limb
[00:14:28.03]based on the origin of a novel,
[00:14:30.08] late phase pattern of Hox expression?
[00:14:32.13] How did this come about?
[00:14:33.27] Is this something we see in fish?
[00:14:35.13] Is this something that comes about with tetrapods?
[00:14:37.06] How is it assembled over evolutionary time?
[00:14:39.23] If you take creatures like Tiktaalik at their word,
[00:14:41.24]the fossils,
[00:14:43.05]it would suggest that perhaps late phase expression
[00:14:44.28]already existed in fish fins,
[00:14:46.26]and maybe it's doing something else.
[00:14:48.15] Let's look at that.
[00:14:50.12]So, the question is really,
[00:14:52.24]when did late phase Hox expression come about?
[00:14:54.08] Did it come about... is it unique to tetrapods
[00:14:57.15]or is it something that we see, primitively, in fish.
[00:15:00.18] So, one of my graduate student, Marcus Davis,
[00:15:02.27] started this quest to understand
[00:15:05.05]patterns of activity of Hox genes in limbs and fins,
[00:15:07.18]and he started by looking at paddlefish,
[00:15:09.27]and you wonder, why paddlefish?
[00:15:11.03]Well, here's a paddlefish.
[00:15:12.17] Paddlefish, it turns out... you can get a lot of embryos of these things
[00:15:15.01]and they have big, fleshy fins.
[00:15:17.23]As you can see in blue, here,
[00:15:19.19]this is the cartilage of the fin.
[00:15:21.12] It's big, fleshy cartilage,
[00:15:22.25]so these are really relatively easy to analyze.
[00:15:26.04]Furthermore, these critters
[00:15:29.06] have a phylogenetic position that's very relevant.
[00:15:31.22] They're very basal ray-finned fish,
[00:15:33.25]so they're sort of close to the branch point
[00:15:36.02]of creatures like Tiktaalik,
[00:15:37.09]so it gives us a window into that.
[00:15:38.28]So, in looking at Hox expression,
[00:15:41.12]Marcus found looking at early expression,
[00:15:42.12]he found they have an early phase pattern of Hox expression.
[00:15:44.19] If you look later on,
[00:15:46.05]they have a late phase pattern of Hox expression.
[00:15:47.29] So, it really does appear they have
[00:15:49.22]two phases of Hox expression,
[00:15:51.01]and that late phase Hox expression
[00:15:52.26]correlates to just like a distal strip of cells
[00:15:55.03]that you see in the distal terminus of the appendage.
[00:15:59.13] The real question here is
[00:16:01.07]if we look at these two phases of Hox expression...
[00:16:03.15]if you look at a mouse,
[00:16:06.12]early phase Hox expression is one
[side] of the chromosome,
[00:16:08.23] on the telomeric phase of the chromosome,
[00:16:11.04] and late phase expression,
[00:16:12.23]that expression that's driven in the wrists and digits and so forth,
[00:16:17.27] is on the centromeric side of the chromosome.
[00:16:19.27]So there's a real structural organization
[00:16:21.21] to the enhancers and regulatory apparatus,
[00:16:24.05]the architecture,
[00:16:25.20]that drives these patterns of late and early phase activity.
[00:16:28.21] And this is well known from mouse
[00:16:30.21]from the work of Denis Duboule's laboratory.
[00:16:32.09] So what we thought we would ask is,
[00:16:33.23]how is this pattern of regulation generated?
[00:16:37.11] Is it present in fish and what is it doing?
[00:16:39.17]And the problem is we don't know much about fish,
[00:16:42.02]so we really had to assemble those data.
[00:16:46.01]But here's the problem:
[00:16:47.24]if you look at late phase expression,
[00:16:50.27] the potential in some of these enhancers
[00:16:52.22]that are present in fish,
[00:16:55.14]you actually have some of the late phase enhancers,
[00:16:57.12]such as this one here, CsB,
[00:16:59.15]and it turns out if you make a reporter of the fish elements,
[00:17:02.10] say from fugu,
[00:17:04.08]and put it in a mouse reporter,
[00:17:05.20]you don't get any activity in the limb.
[00:17:07.16] So the earliest analyses seem to suggest
[00:17:11.28]that late phase enhancers, regulatory apparatus,
[00:17:14.24]are present in fish genomes,
[00:17:18.23]but they're not active in limbs,
[00:17:20.26] that they're not capable of driving late phase expression.
[00:17:22.22]This kind of analysis suggested
[00:17:25.02] that late phase expression is unique to digits,
[00:17:28.24] unique to tetrapods,
[00:17:30.08]and that regulatory apparatus is there,
[00:17:32.18]but not functional in the same way.
[00:17:35.15]Well, it turns out if you look at this,
[00:17:37.00] it seems to be maybe we're not
[00:17:39.24]relying on the right animals for comparison.
[00:17:41.09]So, let's take this area here.
[00:17:44.05] Here is a chromosome,
[00:17:45.17]you can see the Hox cluster on the left,
[00:17:47.01]and on the right, shown in green,
[00:17:48.08] are early phase enhancers.
[00:17:50.12]If you look at a VISTA plot of this enhancer,
[00:17:55.13]comparing human through fish,
[00:17:58.01]through zebrafish and pufferfish,
[00:18:00.27]and you compare the similarities of these regions,
[00:18:02.17]what you'll see is...
[00:18:04.04]you have curves on the top, the human and the chicken,
[00:18:05.29]which suggest they are very similar,
[00:18:07.25] that they have this early phase enhancer.
[00:18:10.03]But if you look at the zebrafish and the pufferfish,
[00:18:11.29]no lines whatsoever --
[00:18:13.17]there's no conservation at all.
[00:18:15.08]So, this kind of conservation analysis
[00:18:17.29]would suggest that these enhancers aren't even present
[00:18:20.15] in fish fins.
[00:18:21.19] Well, it turns out we might not be comparing the right animals,
[00:18:24.14]and the reason for this is that
[00:18:26.26]fish have a whole genome duplication.
[00:18:28.15]And there are three people from my lab
[00:18:29.29]who have been working on this particular problem:
[00:18:31.17]Andrew Gehrke is a graduate student,
[00:18:33.20]our colleague and collaborator José Luis Gómez-Skarmeta,
[00:18:36.05]and Tetsuya Nakamura, a postdoc in my laboratory.
[00:18:40.09]And they've been interested in this whole genome duplication
[00:18:42.28] as perhaps a reason
[00:18:45.19]for maybe why we're not seeing these enhancers
[00:18:47.08] in certain kinds of fish.
[00:18:49.08]Look at it this way:
[00:18:50.26]if we look at the Hox clusters,
[00:18:52.11] in humans there are four Hox clusters,
[00:18:54.01]you can see it in the middle here...
[00:18:55.15] if you look at basal creatures such as Amphioxus or jawless fish,
[00:18:57.24]what you'll see is there is a set up duplications
[00:19:00.26] that go from the single Hox cluster shown in Amphioxus
[00:19:04.21] to the four clusters that are shown in humans.
[00:19:08.01] But if we look at living fish,
[00:19:09.13] the ones that have been the basis for the comparisons
[00:19:11.01]we've already talked about,
[00:19:12.21]you can see they're taken this duplication one step further.
[00:19:15.13] Zebrafish have eight of these clusters,
[00:19:18.05] in fact even salmon have sixteen of them.
[00:19:20.05]So how do you know what to compare?
[00:19:21.19]Maybe functions have been shuffled between them.
[00:19:23.24] So the idea of Andrew in the laboratory is, maybe,
[00:19:27.07]what if we took a fish that didn't have this whole genome duplication,
[00:19:31.16]say a gar,
[00:19:33.13]and use that as the basis of comparison?
[00:19:34.18] Maybe having the right fish system
[00:19:36.26]would allow us to pick up these enhancers
[00:19:38.28]which we're not seeing in other fish.
[00:19:42.17] The good news for us is working with John Postlethwait
[00:19:44.26]and Ingo Braasch from Oregon,
[00:19:46.14] the genome of a spotted gar is now available,
[00:19:49.10]and this was very fortunate for us,
[00:19:50.21]we were able to apply this genome.
[00:19:52.14]So, when we take the gar and put it in this comparison...
[00:19:55.00]you recall, what we showed before is...
[00:19:56.09]here is the human and the chicken
[00:19:58.24]shown on the baseline comparison to a mouse.
[00:20:00.18]You can see there's lots of similarity.
[00:20:02.05]Remember, the take-home message before was zebrafish and pufferfish
[00:20:04.27]don't show any similarity.
[00:20:06.08]When we take the gar as a unit of comparison,
[00:20:09.02]the story changes dramatically.
[00:20:10.18]Here, you have the human and the chicken,
[00:20:12.12]but look, the gar now shows this enhancer peak conserved,
[00:20:15.07]and even having the gar as an intermediate taxa
[00:20:17.07]enabled us to pull out small peaks
[00:20:19.17] for both the pufferfish and the zebrafish,
[00:20:21.27] which were invisible to us before.
[00:20:24.21]Now the question is,
[00:20:26.14] is this chromatin accessible
[00:20:28.13] at the right stage of development?
[00:20:29.15] Are these functioning like real enhancers?
[00:20:31.10]For that we used a new technique
[00:20:33.19]known as ATAC-seq,
[00:20:35.24]which shows us the accessibility of the chromatin
[00:20:37.28] at the right stage.
[00:20:39.01] We can ask the question, by looking at this,
[00:20:40.23]is this enhancer, CNS65, accessible?
[00:20:43.13] And you can see here those large peaks
[00:20:45.18]you see in whole-body,
[00:20:47.21]24-hours post-fertilization,
[00:20:49.15]they show that that chromatin is accessible,
[00:20:52.04] functioning likely as an enhancer.
[00:20:54.08]Now, when we take that enhancer region
[00:20:57.08]and we put it in a fish with the reporter,
[00:20:59.15]here's how it drives expression.
[00:21:01.05] It drives expression throughout the fin
[00:21:03.19] in 31 hours post-fertilization
[00:21:06.21]and then knocks out at 60 hours post-fertilization.
[00:21:10.05]When we take the fish element
[00:21:11.21]and put it in a mouse
[00:21:13.29] we get the same pattern.
[00:21:15.07] Early in mouse limb development
[00:21:17.03]it's driving expression throughout the forelimb,
[00:21:18.28] and in late development
[00:21:21.08] it begins to knock out in the area
[00:21:23.18] that will form the distal part of the appendage.
[00:21:25.18] So, the fish element in mouse
[00:21:27.28] is functioning just as it should for an early phase enhancer.
[00:21:30.26]So, this is a case where having the right model organism
[00:21:33.08] allowed us to find an enhancer
[00:21:36.21]present in fish that has a conserved function with mouse.
[00:21:39.01]Now, the real question we're interested in is
[00:21:41.02] not just these early phase telomeric enhancers;
[00:21:43.12]what we're interested in
[00:21:45.09] is those centromeric enhancers all the way on the right,
[00:21:46.28] because these are the ones that are driving digit expression.
[00:21:49.27]So here we're asking the question,
[00:21:51.13]do fish have the genetic apparatus
[00:21:54.10]that drives Hox activity
[00:21:56.10]which drives the formation of digits?
[00:21:59.22]And here's the whole Hox cluster...
[00:22:01.08] just to make a long story short,
[00:22:02.25]there's the Hox cluster itself,
[00:22:04.16] these are the early phase enhancers, here,
[00:22:07.00]on the left in yellow are the late phase enhancers.
[00:22:09.20]And what I just showed you is
[00:22:11.24]early phase enhancers are present in fish,
[00:22:13.17] this is what I just showed you,
[00:22:14.28]and you can see they report both in mouse and in gar,
[00:22:17.16] and in very similar ways,
[00:22:19.05]and they function as early phase enhancers...
[00:22:20.27]you'll notice how the expression is knocked down
[00:22:23.17]in the distal fin.
[00:22:25.08] Now when we look at the late phase enhancers,
[00:22:27.26] indeed they are present in fish fins
[00:22:30.06]when you add the gar to the comparison.
[00:22:33.02]They report in very similar ways in mouse and in gar,
[00:22:36.14]you'll see the expression activity driven
[00:22:38.17]by the gar element in a mouse,
[00:22:39.22] it's very similar to that
[00:22:41.20]driven by a mouse element in a mouse.
[00:22:43.17]And indeed, when we look at their expression in the fin,
[00:22:46.02]what they do is they drive expression
[00:22:47.22]across the entire fin in early development,
[00:22:49.15]but only in the distal fin in later development,
[00:22:51.29]and the same is true for other late phase enhancers
[00:22:54.24] seen in mouse.
[00:22:57.01] They drive activity both endogenously in mouse,
[00:22:59.26]but the gar element also drives activity within the mouse,
[00:23:03.04] and you can see all the way on the left, here,
[00:23:05.12]just like a late phase enhancer should,
[00:23:06.26] it drives activity throughout the fin in early development
[00:23:09.12]and just in a distal strip of tissue in late development.
[00:23:13.20]What's interesting here is when we take the gar
[00:23:15.29]and put it in mouse
[00:23:16.27] and the endogenous mouse,
[00:23:19.06]they are very similar.
[00:23:20.13]Yet the zebrafish, an animal with that duplicated genome,
[00:23:22.22]barely even reports in the mouse genome.
[00:23:25.06]So, this is a case to show,
[00:23:27.25]when you have the right genetic model,
[00:23:30.16] the right genomic model,
[00:23:32.07]you can see hidden similarities
[00:23:34.10] that would be hidden to you otherwise.
[00:23:36.04]The zebrafish doesn't report easily in mouse,
[00:23:37.29]probably because it has that duplicated genome,
[00:23:40.06]whereas the gar, which has the unduplicated genome,
[00:23:42.13] reports very much like a mouse,
[00:23:44.11] it behaves very much like a mouse.
[00:23:46.21]So, just to give you a sense, looking at other Hox genes,
[00:23:48.10]I just want to show this for one simple point...
[00:23:50.10]when you look at the endogenous activity of these enhancers
[00:23:54.27]in a fin,
[00:23:56.13]what you'll notice is they drive expression
[00:23:58.10]in a distal strip of tissue,
[00:24:00.08]just like fish,
[00:24:01.20]late phase expression of Hox genes.
[00:24:03.03]These same elements in mouse
[00:24:04.28]drive distal expression across the mouse paddle,
[00:24:08.28]which becomes the digits and the wrist.
[00:24:11.03]So, the idea here is a developmental
[00:24:13.12]and genomic equivalency
[00:24:15.17]between that distal strip of tissue you see on the right of the fish fin
[00:24:17.27]and the entire distal paddle of a mouse limb.
[00:24:21.10] So, this leads us to the evolutionary comparison,
[00:24:24.04]supported by Tiktaalik, fossils like Tiktaalik,
[00:24:26.25]and supported by the developmental biology,
[00:24:29.09] is that fish indeed do have wrists,
[00:24:31.27]and if we take the developmental data at face value,
[00:24:34.04] it seems like the distal region of a fish fin,
[00:24:36.02]which consists of those little blobs shown in yellow, on the left,
[00:24:39.27]correspond to the wrists of humans.
[00:24:44.24] So, the take-home message here is
[00:24:47.08]we can leverage multiple lines of data
[00:24:49.12]to understand evolutionary history.
[00:24:51.04] Look, I'm a paleontologist,
[00:24:52.23] I don't find enhancers buried in rocks,
[00:24:54.15] but what I have is the means
[00:24:56.29] to compare the enhancers of living creatures
[00:24:59.20] that are separated by huge phylogenetic distances.
[00:25:02.17] The way we do it is first start with fossils
[00:25:04.29] to bridge the gaps
[00:25:06.18]and then devise experiments
[00:25:08.06]which help really bridge those gap
[00:25:10.05]s in a mechanistic way.
[00:25:11.13]So it's really an exciting time for science
[00:25:13.08]because we can begin to analyze evolutionary transformations
[00:25:16.17]using both genetic and molecular data,
[00:25:18.27]as well as classic paleontological data.
[00:25:20.27]To do an analysis like this takes amazingly talented people,
[00:25:23.24] including the artist who drew this lovely diagram,
[00:25:26.22] as well as my members of my laboratory,
[00:25:29.20]my good colleagues,
[00:25:31.03]who have co-led the Tiktaalik expeditions with me,
[00:25:33.21]the Inuit community and Canadian government,
[00:25:35.21]which has supported our work for several decades,
[00:25:39.04]my molecular colleagues
[00:25:40.27]and the colleagues who have provided access
[00:25:42.17]to the gar genome,
[00:25:44.14] and of course the funders of our work.
[00:25:46.07]Thank you very much.